Amperometric enzyme sequence eletrodes for cholesterol

Abstract

Abstract An enzyme electrode for the determination of free cholesterol in serum samples was developed. The Michaelis constants of soluble and immobilized cholesterol oxidase (COD) used for the sensor were 2.2×10 −5 and 2×10 −4 mol/dm 3 respectively, and 6×10 −4 mol/dm 3 for the COD electrode. The immobilization of COD on modified 2-hydroxyethylmethacrylate gel resulted in an activity of 0.3–0.45 Unit/g (U/g). The principle of the assay with the COD-enzyme electrode is the anodic oxidation of H 2 O 2 produced during enzymatic cholesterol oxidation. The linear range of concentration was 0.4–12 m M for the cholesterol sample concentration (20 mm 3 sample volume), with a response time of 0.5 and 2 min for the kinetic and steady-state methods, respectively. The described immobilization technique was applied to covalent binding of cholesterol ester hydrolase (CEH) and COD. This preparation was used in direct spatial contact with a polarographic system forming the enzyme sequence electrode for total cholesterol assay, thus representing the first bienzyme electrode for total cholesterol determination. The COD electrode was combined with the HRP reaction and other electron mediators, e.g. ferrocyanide.